Science - USA (2018-12-21)

Ruscettiet al.,Science 362 , 1416–1422 (2018) 21 December 2018 4of7

A C

B

Vehicle

Trametinib

Palbociclib

Combo

*** **

**

* **

**

Change in tumor cells (%)

• 
  
  
  
  • 
    
  
  
  
  


• 
  
  
  
  • 
    
    
    
    • 
      
    
    
    
    
  
  
  
  

+ +

+

Palbociclib

Trametinib - +

• 
  
  
  
  • 
    
    
    
    • 
      
    
    
    
    
  
  
  
  

+ +

+

A549 H460

row max

row min

IL-15 IL-18

IFN-IFN 2

CCL5

CCL2

CCL3

VEGF

PDGF

G-CSF

GM-CSF

IL-7

IL-4

IL-2

IL-5

sCD40L

TNF-

IL-12p70

TGF-

FGF2 EGF

FLT-3L

CCL22

CCL11

CX3CL1

IL-8

IL-1

IL-10

IL-6 IL-12p40

CCL7

CCL4

CXCL10

PDGF

CXCL1

IL-17AIL-1RA

IL-1

IL-3

TNF-

IL-9

IL-13

H2030

HCC15

A549 +

+

• 
  

RB p53

+

• 
  


• 
  

D

SASP

Cell Cycle

E

G *

SA-

-gal (%)

0

5

10

15

20

25

Isotype NK1.1

Trametinib

*** **

**

**

Vehicle Palbo

Combo + IsotypeCombo + NK1.1

50

0

-50

-100

-150

Change in tumor area (%) -200

H

ICAM-1-FITC

Vehicle Tramet Palbo Combo

Isotype NK1.1

H2030 A549

-75

-50

-25

0

25

rametinib

oa

row

min

IL-15

IL-12p40

IFN

IL-6

CCL5

CCL2

CCL4

PDGFFGF2

GM-CSF

IL-13

EGF

CXCL10

CCL11

IL-4

IL-9

IL-2

IL-5

TGF-

CCL3

VEGF

G-CSF FLT-3L

CXCL1

IL-7

IL-1

CCL22

CX3CL1

CCL7

PDGF

IL-1RA

TNF-

IL-17A

IL-8

IFN- 2

IL-10

sCD40L

IL-1

IL-3

IL-12p70

IL-18

TNF-

MFI

Vehicle Trametinib Palbociclib Combination

0

10

20

30

40

50

60

70

80

A549

H2030

H460

SA-

-gal

+ (%)

F

ICAM-1 ULBP2/5/6 MICA

0

500

1000

1500

2000

5000

10000

15000

20000 Vehicle

Trametinib

Palbociclib

Combo

****

**

***

****

****

****

***

**n.s.

0102 103 104 105

C P T V C P T V

denia

tsnu

α

(^1) -MAC
I
S100A11TNFAIP2CYP1B1MAP1LC3BSAT1
OPTNTNFAIP3CDKN1AEPS8SQSTM1
NPC1CCND1RNF149CCNE1RAB27B
ORC6WEE1IQGAP3NEURL1BID1
PIRCOMMD4IMPA2RAD51AP1NUCKS1
RHNO1H2AFVNUSAP1DUTFAM111B
CENPMRNASEH2APOLE2HMGB3DEPDC1B
NUF2FAM83DCCNB1AURKAPLK1
SGOL2UBE2CCDC20CHEK1CTDSP1
ASF1BFBXO5MCM6HELLSBUB1B
TK1CDK2DEKUSP1CDK1
CDCA3CCNB2NCAPD2KIF2CFOXM1
BIRC5AURKBH2AFXCENPACCNA2
NASPKIF11TOP2ATMPOHMMR
MKI67PRC1LMNB1SMC4DLGAP5
TYMSHMGB2SPC24CDKN3KIF20A
SPC25STMN1MYBL2H1FXHMGN2
EZH2CDK4MCM3EXO1DNMT1
HMGA1MTL1CAMSTXBP1GM2AND2
CSTBRHOBKRT8MUC1DDR1
CLUGAS6IL6NFKB2CX3CL1
CCL2NFE2L2SERPINE1CXCL11ACVRL1
PVRCD82PLAUIL15SGMS2
AT P 2 C 1CD40BST2ITGA5HIF1A
ATP2A2CSF1IGFBP7KLF6IFNAR1
PSEN1TAPBPIRF1IL1R1NAMPT
CXCL10IL15RAIFNGR1TNFRSF9HLAC
HLABHLAATAP1ATF3IFIT2
GADD45ARCAN1FOSL2BIRC2NINJ1
PNRC1KDM6BDNAJB4STAT1ISG15
XAF1MX1IFIT3B2MSTAT3
V
T
P
C
2
1
0
1
2
H2030 H460 A549
Fig. 2. Senescence and SASP induction after combination MEK and
CDK4/6 inhibitor therapy induces NK cell immune surveillance.
(A) Quantification of SA-b-gal+cells in human KRAS-mutant lung tumor
cell lines after 8-day treatment with trametinib (25 nM) and/or
palbociclib (500 nM). Mean of three biological replicates is plotted.
(B) Clonogenic assay of human KRAS-mutant lung cancer cells replated
in the absence of drugs after 8-day pretreatment as in (A); representa-
tive of three biological replicates.RBandp53genomic status is indicated
on right. (C) Heat map of senescence-associated cell cycle and SASP
gene expression in human KRAS-mutant lung cancer cell lines after
treatment as in (A), as assessed by RNA-seq. Two biological replicates
per cell line are shown. C, combination; P, palbociclib; T, trametinib;
V, vehicle. (D) Heat map of cytokine array results from human
KRAS-mutant lung tumor cells treated with trametinib (25 nM) and/or
palbociclib (500 nM) for 8 days. Data are presented as mean of three
biological replicates. (E) Flow cytometry analysis of ICAM-1 expression
and levels of other NK cell–activating ligands after 8-day treatment of A549
lung tumor cells as described in (A). Quantification of mean fluorescence
intensity (MFI) from three biological replicates is shown on the right.
(F) Quantification of NK cell cytotoxicity (by live cell imaging) after
pretreatment of A549 and H2030 lung tumor cell lines with indicated
drugs for 8 days and coculturing with the YT NK cell line at a 10:1 effector
to target cell (E:T) ratio for 20hours in the presence or absence of
indicated drugs. Change in tumor cells is normalized to control
wells lacking NK cells. Mean of three biological replicates is plotted.
(G) Representative SA-b-gal staining of KP transplant lung tumors after
1-week treatment with combined trametinib (1 mg/kg body weight)
and palbociclib (100 mg/kg body weight) and either an isotype control
antibody (C1.18.4) or NK1.1 depleting antibody (PK136) (scale bar,
50 mm). Quantification shown on right (n= 3 mice per group). (H)Effect
of 1-week treatment as in (G) on lung tumor burden (relative to vehicle)
(n= 5 mice per group). (E, F, and H) One-way ANOVA. (G) Unpaired
two-tailedttest. Error bars, mean ± SEM. P<0.05,P< 0.01, P<
0.001, ****P< 0.0001.
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